The present invention relates to the technical implementation of a method of super-resolution localization microscopy. Such methods are characterized in that during the acquisition of an image, only a few object points emit light simultaneously at a given time. If these points have a spacing significantly greater than the optical resolution, then the positions of these points can be determined by mathematical centroid-determination methods with an accuracy higher than the optical resolution of the objective used. When many of these individual images of different object points are summed, it is finally possible to create a super-resolution image of the entire object. Known variants of such methods differ in the way in which they ensure that only a few object points emit light simultaneously. Examples of names of such methods are PALM, STORM and GSDIM. The latter method will be briefly described hereinafter.
GSDIM stands for “Ground State Depletion followed by Individual Molecule return”. This method uses fluorophores that have triplet states (also “dark states”). Initially, a dye molecule is transferred from the ground state to an excited state by irradiation of adequate laser power. A transition occurs from the excited state to the triplet state, the triplet state having a far longer lifetime (about 100 ms) than the excited singlet state (only about 3 ns), so that given sufficient light intensity, the molecules accumulate in the triplet state. The molecules return from the triplet state to the ground state, producing spontaneous emission. Individual images of these spontaneous emissions are recorded, and the positions of the spontaneous emissions can be determined by the aforementioned mathematical methods with an accuracy higher than the optical resolution. The achievable resolution is between 20 and 25 nm, generally less than 60 nm. However, the achievable resolution also depends on the laser power and the resolution of the camera used.
The main technical requirement for implementing GSDIM is the fluorescence excitation of the specimen with high-power light. This can be accomplished through suitable laser illumination which may, in turn, be provided by a Total Internal Reflection Fluorescence Microscope (TIRF) illumination apparatus, which focuses the laser beam into the pupil of the objective (back focal plane of the objective). This has the advantage of providing homogeneous illumination of the specimen and, at the same time, high depth of focus of the illumination. In order to optimally use the power of the laser, the cross section of the laser beam is reduced to such an extent that the illuminated specimen field is only one-fourth as compared to TIRF illumination (for example, the diameter of the illuminated specimen field is about 60 μm when using a 100× magnification objective, while in TIRF illumination, this diameter is about 250 μm).
The method of switching between the TIRF illumination and illumination for GSDIM will be illustrated below with reference to FIGS. 1a and 1b. 
Following are some initial observations on TIRF microscopy. Typically, an inverse light microscope having an oil-immersion objective with a high numerical aperture is used to achieve the flat angle of incidence required for total reflection. The total reflection occurs at the interface between the cover slip and the specimen. A so-called evanescent field is formed in the region behind the cover slip. The intensity of this field decreases exponentially into the specimen. For visible light, the penetration depth is typically 100 to 200 nm. If fluorescent molecules capable of absorbing the irradiated wavelength of light are present in this region, these molecules can then be excited to emit fluorescent light. Since the observable layer in the specimen is only 100 to 200 nm thick, it is possible to achieve a significantly higher resolution along the optical axis than with normal fluorescence microscopy (layer regions of typically 500 nm). In TIRF microscopy, the excitation light is coupled in at the edge of the objective, so that it strikes the cover slip at the required flat angle.
FIG. 1a schematically shows a typical TIRF illumination with an illumination beam 16: The light (represented by dashed lines) originating from a light source 18 (typically a laser) is collimated by collimating optics 17. The collimated light is focused by a scanning eyepiece 13. Transporting optics 14 image the focus onto back focal plane 6 of objective 7, the light previously being reflected by splitting mirror 19. The splitting mirror may provide, for example, physical separation, geometrical separation, color separation, or polarization separation. Transporting optics 14 are needed due to the mechanical characteristics of a microscope stand, because scanning eyepiece 13 cannot be mounted close enough to objective 7 to be able to focus directly onto its back focal plane 6. To enable the use of objectives which have their back focal planes in different positions, scanning eyepiece 13 is focusable (indicated by an arrow). The laser beam focused into back focal plane 6 of objective 7 is projected by the objective as a parallel beam into the space of specimen 8 (object space). This parallel beam illuminates a circle having a diameter Da (typically about 250 μm) in specimen 8.
For TIRF microscopy, it is crucial that scanning mirror 12 (shown schematically) be tiltable. By tilting scanning mirror 12 (indicated by two arrows), the focus is moved in back focal plane 6 perpendicularly to the optical axis of the objective. As a result, the laser beam passes through specimen 8 at an angle that is dependent on the tilt of scanning mirror 12. When this angle is greater than the angle of total reflection between the cover slip and the specimen medium, then one speaks of TIRF microscopy, as explained above.
The specimen illuminated in this way then emits fluorescent light (represented by solid lines). This light passes through beam splitter 19 and forms an image 22 through a tube lens 21.
FIG. 1b shows a typical illumination for a GSDIM method. What is crucial here is a high laser power per illuminated specimen area. Consequently, it is required that the laser power act on a greatly reduced specimen diameter Db (typically about 60 μm). This can be accomplished by modifying the TIRF illumination described with reference to FIG. 1a in a manner that will allow switching between TIRF and GSDIM illumination. In the TIRF illumination apparatus described above, this is achieved by inserting a telescope 11 (represented by dashed lines) into the optical path to reduce the beam diameter. In this way, the circle illuminated in the specimen is reduced to a diameter Db (here about 60 μm), as a result of which the power density is increased by a factor of about 18. Typically, the laser beam strikes specimen 8 at normal incidence. In rare cases, observation is performed with scanning mirror 12 in tilted position. For further details, reference is made to the explanations given with reference to FIG. 1a. 
The above-described known approach of switching from TIRF illumination to GSDIM illumination has the advantage of being relatively easy to accomplish by inserting a telescope, but it also has the following disadvantages: GSDIM microscopy is very rarely performed under TIRF conditions; i.e., with scanning mirror 12 in tilted position. What is even more important is that TIRF itself is a very rarely used application, so that it appears to make little sense to combine it with GSDIM microscopy. Consequently, most of the users interested in localization microscopy are forced to make a purchase that includes a complex TIRF illumination apparatus which they will rarely use.
Another application is confocal microscopy. In this regard, reference is made to the extensive prior art. Confocal microscopy, unlike conventional light microscopy (also “wide-field microscopy”), does not illuminate the entire specimen, but only a portion or point of the specimen at any point in time. The specimen is illuminated and scanned point by point through the microscope objective using a suitably focused laser or a point light source. Thus, it is possible to successively measure the intensities of the reflected light at all scanned positions of the specimen. Subsequently, an image of the specimen is constructed from the measured light intensities. Due to their high axial resolution, confocal microscopes enable acquisition of a large number of such images at different focal planes, and thus generation of a sharp three-dimensional image. To this end, the excitation light of the laser or point light source is focused into different focal planes in the specimen. The light reflected from the specimen is imaged onto a pinhole, generally through the same objective through which it is focused onto the specimen, and passes through the pinhole to a detector. The excitation focus and the detection focus are confocal to each other, i.e., coincide. From there, the name “confocal microscopy”. Light from planes outside the focal plane cannot pass through the pinhole to the detector. By scanning the specimen in the x-y direction; i.e., in a plane perpendicular to the optical axis of the objective (z-direction), it is possible to obtain the image of the specimen in the selected focal plane (z=z0).
U.S. Pat. No. 7,187,494 B2 describes a microscope system which allows switching from TIRF microscopy to confocal microscopy. FIG. 6 of this document illustrates a typical configuration of a laser scanning microscope for confocal microscopy. FIG. 7 of this document depicts the typical configuration of a TIRF microscope. Based on a combination of a confocal microscope and a TIRF microscope, as shown in FIG. 8 of that document, this U.S. patent deals with an alternative solution for such a combination, which includes the use of optics that can be inserted into the optical path of the confocal microscope to focus the laser light into the back focal plane of the objective, with the chief rays being parallel to the optical axis at the position of the back focal plane. The latter requirement is necessary for TIRF illumination. As an alternative embodiment of the aforementioned combination, this patent publication describes the use of a complete, folded optical path to allow switching between TIRF and confocal microscopy.
U.S. Pat. No. 7,551,351 B2 describes a microscope with TIRF illumination for optically manipulating a specimen. This document proposes a microscope system whose illumination can be switched between TIRF illumination and manipulation laser illumination. The manipulation laser is used for bleaching, for marking purposes and/or for microdissection. Switching between TIRF illumination and manipulation laser illumination is accomplished either using a mirror which can be inserted into the optical path of the TIRF laser, or using an activatable lens.
German Patent Publication DE 199 012 19 A1 discloses an optical arrangement disposed in the illumination beam path of a confocal laser microscope and adapted to provide optimal illumination while reducing loss of excitation light. To this end, a lens and downstream varifocal optics are disposed downstream of the laser. This arrangement is used to expand the narrow cross section of the parallel laser beam. The beam expanded in this way is directed onto a scanning mirror and reflected by it toward the objective. The beam passing through the entrance pupil of the objective is focused by the objective onto the object. Using the aforementioned varifocal lens, the diameter of the illumination beam can be adjusted, with greater or lesser accuracy, to the entrance pupil of the objective used in order to prevent loss of light.
It is an object of the present invention to provide a (second) non-confocal illumination which is particularly suitable for localization microscopy, in particular for a GSDIM method, and which can be used independently of a TIRF illumination. In particular, such an illumination is intended to be switchable to a (first) illumination that is suitable for confocal microscopy.